Method for producing 2-keto-lgulonic acid



United States Patent 3,234,105 METHOD FOR PRODUCING Z-KETO-L- GULONICACID Kazuwo Motizuki, Isoshi, Takarazuka, Toshihiko Kanzaki,

The present invention relates to a method for producing Z-keto-L-gulonicacid which serves as an intermediate for the production of thephysiologically important L-ascorbic acid, well known as vitamin C, andmore particularly to the 2-keto-L-gulonic acid production whichcomprises transforming sorbitol to 2-keto-L-gulonic acid by the actionof the enzyme system of a microorganism selected from the genusPseudomonas or the genus Acetobacter.

Z-keto-L-gulonic acid may be produced from D-glucose through sorbitol,L-sorbose, diacetone sorbose and diacetone 2-keto-L-gulonic acid as theintermediates by the Reichstein method, or through 5-keto-D-gluconicacid and L-id onic acid by the Gray method. These methods, however, mustpass through several reaction steps. This renders the above processesunsatisfactory from industrial point of view as these are difi'icult inworking as a continuous process.

One object of the present invention is to provide a process wherein theproduction of 2-keto-L-gulonic acid can be effected without the saiddisadvantage. In other words, an object of the invention lies in theembodiment of a process which comprises transforming sorbitol to 2-keto-L-gulonic acid through only one step by the action of the microbialenzyme system.

Another object of the present invention is to providemicroorganismswhich produce the enzyme system capable of transformingsorbitol to 2-keto-L-gulonic acid.

Still another object of the present invention is to provide a method forthe production of ascorbic acid by subjecting the ester prepared from2-keto-L-gulonic acid to enolization and lactonization, which results ina good yield.

Pseudomonas and Acetobacter, in the present specification, are the namesof genera belonging to the family Pseudomonadaceae and the orderPseudomonadales, the classification being in accordance with that inBergeys Manual of Determinative Bacteriology, seventh edition, 1957, byRobert S. Breed, E. G. D. Murray and Nathan R. Smith, published by TheWilliams & Wilkins Company, United States of America.

The microorganisms to be employed for the production of the enzymesystem capable of transforming sorbitol to 2-keto-L-gulonic acid arethose belonging to genera Pseudomonas and Acetobacter. Morespecifically, some examples are mentioned in the following:

Acetobacter melanogenum, ATCC 15163 (IFO 3292) Acetobacter sp., ATCC15164 Acetobacter rubiginosus, IFO 3243 Acetobacter suboxydans, NRRLB-72 Acetobacter gluconicus, ATCC 9324 Acetobacter xylz'num, ATCC 10245Acetobacter albidus, IFO 3250 Acetobacter albidus, IFO 3251 Acetobacteralbidus, IFO 3253 Acetobacter subo'xydans, IFO 3255 Acetobacterindustrius, IFO 3260 Bacterium orleanense, IFO 3259 3,234,105 PatentedFeb. 8, 1966 "ice Acetobacter cerinus, IFO 3263 Acetobacter cerinus, IFO3264 Acetobacter cerinus, IFO 3265 Acetobacter cerinus, IFO 3266Acetobacter cerinus var. ammoniacunIFO 3267 Acetobacter cerinus var.ammoniacus, IFO 3269 Acetobacter cerinus, IFO 3268 Pseudomonasstriafaciens, IFO 3309 Pseudomonas coronafaciens, IFO 3310 Pseudomonassp., ATTC 15165 No'rn.Baaterium ofleanense belongs to the genusAcetobacter according to the classification system of Bergeys Manual ofDeterminative Bacteriology.

No'rn.* denotes new strain.

Numbers and abbreviations attached to the names of microorganisms inthis specification show the respective accession numbers to the strainin Northern Utilization Research Branch, US. Department of Agriculture,Peoria, 111., USA. (NRRL) or American Type Culture Collection,Washington, DC, U.S.A. (ATCC), or Institute for Fermentation, Osaka,Japan (IFO).

Determination of species name is, however, sometimes complicated andentangled. At any rate, the microorganisms capable of producing theenzyme system causing transformation of sorbitol to 2-keto-L-gulonicacid, which belong to the genus Pseudomonas or genus Acetobacteraccording to the classification by Bergeys Manual of DeterminativeBacteriology, are all involved in what is called genus Pseudomonas orgenus Acetobacter in this specification, even if they may come underother generic names in other nomenclatures.

Microorganisms, which are employed in the method of this invention, areall available in known culture collections, such as Centraalbureau voorSchimmelcultures, Holland, or Institute for Fermentation, Osaka, Japan,or Northern Utilization Research Branch, US. Department of Agriculture,Peoria, Illinois, or American Type Culture Collection, Washington, DC,or National Collection of Industrial Bacteria, Teddington, England. Ortheir wild cultures can easily be obtained from flowers, fruits, soil,

etc.

The [above-mentioned microorganisms can be induced to mutants havingsuperior ability relative to the parent wild strains of producing theenzyme system capable'of transforming sorbitol to 2-keto-L-gulonic acid.Such mutation can be caused by treating a wild strain with a mutagensuch as ultraviolet rays irradiation, X-ray irradiation or contact withnitrous acid, or by isolating a clone occurring by spontaneous mutation.These means for inducing the desired mutation on a wild type strain maybe effected in any of the ways per so well known for this purpose by theskilled in the art and which have been described in many publications,for example, Methods in Medical Research, volume 3, edited by R. W.Gerard, published by the Year Book Publishers, Inc., Chicago, U.S.A., in1950, and Nature, volume 183,

1 page 1829 (1959), reported by F. Kaudewitz The characteristics of theaforementioned new strains are shown in the following enumerations:

CHARACTERISTICS OF ACETOBACTER SP. ATCC 15164 Rods, occurring singly orpairs. Sometimes, the rods become of filamentous involution form. Gramnegative non-motile.

Hennebergs lankes, Hayducks and Beijerincks synthetic aqueous media: nogrowth.

Agar slant: glistening, smooth.

Gelatin: liquefied.

Assimilate peptone but not ammonium salts.

Optimum pH for growth: 6.06.4.

Do not oxidize acetic acid.

tolgluconic acid, 2-keto-gluconic acid and 2,5-diketo-gluconic acid.Oxidize mannitol to fructose and further to kojic acid. Form acid frommaltose and ethanol.

CHARACTERTERISTICSOFIPSEUDOMONAS SP. ATCC 15165 Nutrient broth: surfacegrowth none, clouding moderate, flaky sediment leptone water: surfacegrowth none, clouding slight,

, sediment scanty.

lndoln tpr d ed- Hydrogen sulfide produced. Starch nothydrolyzed.

, Nitrites not produced from nitrates.

. iArnmoniam not produced. V Methyl [red positive, Voges-Proskauer testsnegative.

Litmus milk: acidic, coagulate.

,' Catalasez, positive. I Optimum temperature for growth: about 28 C.

Optimum pH for growth: 6.0-6.5.

Acids from arabinose, xylose, glucose, fructose, galactose,

mannose, sucrose, glycerol. No acid from' sorbose, lactose, maltose,treharose, ramnose, starch, inulin, dextrin, mannitol, sorbitol ethanol.

No gas from thesesugars.

Incarrying out the method, of the presentfinvention,

the; following rtwo processes are conveniently employed: (1) Amicroorganism is cultured in a medium con- .taining sorbitolandotherappropriate'nutrients and (2) f i'after cultivation, the. whole?culture, the cells collected fromfth'e culture, or enzyme preparationsobtained from the cells by means of conventional methods inenzymologyare brought into contact 'with sorbitol.

. In .the case of .theprocess (1) described above, the

l microorganism may be cultured in an aqueous medium in aeratedfermentor. ducted at pH values between about 4 to 9, butpreferablybetween about 5 to 8. Apre'ferred temperature is from Theficultivationshould be conabout' to 30 C.,' especially from about to C. While thetime for the cultivation varies with .the kind of fmicroorgani'sms andnutrientrnedia to be used, aboutS to 10 day cultivation may usuallybring most preferable yield. The concentration of 'sorbitol Y in themedium varies 5.- Substancesmentioned above are also used for nutrients.

gram/liter to about 200 grams/liter: The mixed solution 4 microorganismsto be employed, it is preferable tochoose a propermediumcase by case.

In the case of the process (2), the microorganism may;

by cultured under similar conditions to the process (1).

About'l to 2 days culture is usually preferable for obtaining mostadvantageous cells forf'thes produ-ction of 2-keto- L-gulonic acid fromsorbitol. Then; in one case', sorbitol per se or its aqueous solutionis'added intothe cultured medium to make its final conccntrationlfrosnabout 1 may be incubated for about'orre day underlt'he same condi-vtions as, in the case of the preceding cultivation. ,In another case,they cells "may be collected by 'centrifugation from the culture brothand resuspended in an 'aqueous medium bufferized with phosphates,;tris(hydroxymethyl) aminomethane, etc.;at a pHfro mmbout 5 :to 8. Thensorbitol is added in the same way as mentioned above.

The sueceedmg incubation maybe effected under" similar conditions asthose described above. p H

In the method of thef-present invention, the resulting f2-keto-L-gulonic acid 'in the reaction *mixture'need" not be isolated,but: the reaction "mixture can directly be es ter ified, followed byenolizationi and lactonization, 'and can be led to L-ascorbic acid; In"'caseof isola'tingthe objective 2-keto-Legulonicacid"fromithe'reactionmixture,

howeverpthe, isolation may desirably 'be effected as its salt form or byutilizing diifer encesbetween the product and impurities, in suchpropertiesfas' solubility-adsorbability, distribution coefficientbetween two solvents. Use

' of an adsorbent such asiionexchangeresin is onej'ofl-the mostconvenient processes'for' the isolation of the product.

Since the Z-keto-L-igulonic acid'thusobtainedis, general,

notpure, it may be purified by conventionalmethods such;

as recrystallization, and chromatography.

As stated above, 2- keto-L-lgulonic acid canbe'led to. Li-ascorbic acid:by "application of an'y' ohfthe :known,

' methods, if desired. L-ascorbic acidis generally synthesizedbyesterifyin'g Z-keto-L-jgulcinic acid in the presence V of amineralia'cidjsuch assumes acid, hydrochloric acid or strongly acidiccationex hange;resiugasia: catalyst,

followed by enoliging thefester and subsequently l'a ctonizsf e' 01 c pd v V. .7

'The preferred strongly acidic cation exchange resins which can beemployed for esterification of 'Z-keto-L- gulonic acid may be eithersulfo'nated phenolic, or'sulfo- 'natedpolystyrene' resins; the size:offparticles,ithe degree. of cross-linking, andflthe ion exchangecapacity are of little significance in carrying jout the present"invention. Thus, by Way of example, Amberlite =IR-12(),Amberlite withthe kind of microorganisms, but is it generally de- Tsirable to' be,about 1 to 200 grams/liter, most preferably, about 5 to 100 grams/liter.

. Itis usually required that the.culture medium contains such nutrientsfor the microorganism as assimilable car- Mbon sources, digestiblenitrogen sources, and preferably inorganic substances, vitamins, .trace.elements, othergrowth promoting factors, .etc.. Sorbitol per se servesas the" carbon'source, b u-tthefollowing substances may also i be usedas auxiliary carbonsources: starch, cane sugar,

.Tlactose, dextrin, glycerol, maltose, etc. They are employed ataconcentration from about 1 gram/liter to 10 grams/liter. As the nitrogensources there may be used various zorganicior inorganic; substancessuch, as, soybean 1,

meal, meat extracts,'peptone, casein, yeast extracts, corn steep'liquor,urea, nitrates, ammonium salts, etc. As the inorganic nutrients, forexample, potassium phosphates, magnesium sulfate, ferrous and ferricchlorides,

calcium carbonate, etc. areusually employed. As the constituents of themedium'vary' also with the kind of,

Philadelphia, Pennsylvania, 'U.S.A. Dowex 50, Dowex- W (Dow Chem'icaljCo.,' lncf, Midland, Michigan, U.S.A.), Duolite C- 3' (ChemicalProcessaCog Inc}; Redew d City, California, U.S,A.)," andl Diaion' SK-l(Mitsubishi Chemical Industries, Limited, Tokyo, Japan),

are those w'liich canbe advantageously employed fo'r'the purpose stated.

The' resulting ester inthe reaction mixtureheednot be separated; but the"reaction mixture than directly be. subjected to e'noliz'ation'andl'actonization. V For the enoli "z'ation' andlactonizatiom-any of theknow'n'm'ethods are applicable. Arnong'lsuchjm'etho'dsj', there areincluded the method -wl1erein thereactioni product. (ester) is"heated ina neutral solvent, the method whereinfthe reaction product: (ester) isheated inithe presence "of? anion exchange. resin, metalpowder,"sodiumalcoholate;salt of a Weak acid such as sodiumhydrogencarbonate,"sodium carbonate and: calcium carbonate,orthezinethodiwherein the reaction product (esterfis heated in'an'acidic s'olvent such as hydrochloric acid.f"Above,all,ithe methodsin which the reaction p roductf(ester) is'he2itedi in the. presence ofanion exchange 'resinare most preferred,

because by employing suchmethods it is made' possible to effectesterification, process," enolizationprocess and lactonization processin a continuous manner. Among the anionexchange resins which can be usedfor enolization and la-ctonization of 2-keto-L-gulonic acid ester, theremay, for example, be enumerated Amberlite IRA-400, Amberhte IRA-410,Amberlite XE-168, Amberlite IR 45, etc. (Rohm & Haas Co., Inc.,Philadelphia, Pennsylvania, U.S.A.), Dowex-l, and Dowex-2 (Dow ChemicalCo., Inc., Midland, Michigan, U.S.A.).

Incase thus obtained L-ascorbic acid is in a form of salt such as sodiumsalt, calcium salt, etc., it may, if desired, be converted to the freeform by hydrolyzing with an acid, such as hydrochloric acid.

The present invention will now be described in further particularity bymeans of the following illustrative examples. It will be understood, ofcourse, that the invention is not limited to the particular details ofthese examples since they only set forth preferredexemplary' embodimentsof the invention. In these examples, all percentages are on a weightbasis, unless otherwise noted. Temperatures are all uncorrected, andabbreviations g, mg, 1., and ml. mean gram(s), milligram(s), liter(s)and milliliter(-s), respectively.

Example 1 Cells of a selected strain of Acetobacter sp., ATCC 15164,grown on a Sorbitol-yeast extract agar slant, for 2 days at 28 C. weresuspended in sterile water. 1 ml. of this cell suspension was inoculatedinto a 1 I. shake flask containing 150 ml. of the following aqueousmedium, previously sterilized in an autoclave at 15 lbs. per square inchsteam pressure:

- Percent Sorbitol 2.0 Glucose 0.5 Yeast extract 0.5 CaCO 1.0

Elemental analysis for C H O -H O: Calculated: C, 0

34.01%; H, 5.7%. Found: C, 34.74%; H, 5.29%.

:Methy1 ester melting point: 154155 C.

Elemental analysis for C7H120q: Calculated: C,

40.28%; H, 5.62%. Found: C, 40.3%; H, 5.6%.

Example 2 One loopful of cells of a selected strain of Acetobacter sp.,ATCC 15164, were inoculated into a shake flask containing 500 ml. of thefollowing aqueous inoculum medium and incubated at 30 C. with shakingfor 18 hours:

. Percent Sorbitol 2 Glucose 0.5 Yeast extract 0.5

A basal medium was prepared for actual production of 2-keto-L-gulonicacid. This medium had the following composition:

Percent Sorbitol 5.0 Glucose 0.5 Yeast extract 0.5 CaCO 2.0

500 ml. of the incubated inoculum was added to 30 l.

2 keto-L-gulonio acid g/ml.)

, 6 of the above medium in a fermentor having a total volume of 50 l.

Incubation was conducted at 28-29 C. with stirring at the rate of 280r.p.m., with aeration at the rate of 15-24 liters per minute, and withinner pressure at 15-20 lbs. per {square inch. After 150 hoursincubation, yield of the product, 2-keto-L-gulonic acid, attained toabout 500 mg. per ml. of the culture broth. Cells and solid materials inthe culture broth were discarded by centrifugation and about 27 litersof the clear filtrate was obtained. 1 liter of the filtrate wasdecolorized with 50 g. of activated carbon and passed over Amberlite IR-resin and then concentrated in vacuo. The yield of 2- keto-L-gulonicacid was about 4.3 g. per liter of the filtrate.

Example 3 Cells of a selected strain of Acetobacter cerinus, IFO 3266,grown on Sorbitol-yeast extract agar slant, for 2 days at 28 C. weresuspended in 10 ml. of sterile water. 5 ml. of this cell suspension wasinoculated into a one I. shake flask containing ml. of the followingaqueous medium, previously sterilized in an autoclave at 15 lbs. persquare inch steam pressure:

Percent Sorbitol 5 Glucose 0.5 Yeast extract 0.5 CaCO 2.0

Incubation was maintained at 28 C. with shaking at the rate of 225r.p.m. for 10 days. The culture broth was filtered and the filtrate waspassed over Amberlite IR-120 resin. The product, 2-keto L-gulonic acid,was then adsorbed on Amberlite IR-45 resin. After Washing the columnwith water, the product was eluted with 1 N potassium hydroxide and theeluate was concentrated and decolorized with activated carbon. Thedecolorized solution was again passed over Amberlite IR120 resin and thepassed solution was concentrated with flash evaporator. 4.7 g. of thecrude Z-keto-L-gulonic acid was obtained from the broth collected from10 shake flasks.

Example 4 One loopful of cells of a mutant strain of Acetobactersuboxydans, NRRL B-72, were inoculated into a 200 ml. shake flaskcontaining 15 ml. of the following aqueous medium and incubated on arotary shaker at 28 C.:

Percent Sorbitol 2.0 Polypeptone 0.1 Casamino acids 0.25 Glycerol 0.1 KHPO 0.05 MgSO -7H O 0.05

Days s 5 7 Example 5 A mutant strain of Acetobacter suboxydans, NRRLB-72, was rinsed from an agar slant into a 2 I. shake flask'c0ntaining500. ml. of. the following. aqueous me.- dium:

dium inia fermentor having a total volume of 50 1. In 1 cubation wasconductedat 28 Cvwith stirring at the rate of 260 r.p.m. and withaeration at the rate of 30 1. per minute. The progress'of the reactionwas followed 'by taking out a small amount-of the broth and-estirnatingthe 2-keto-L-igulonic acid contentasdescribed in-Example .41

Result obtained: asfollows:

buys. 3 5 7' 10' i 2 keto-'L-gulonic acid g./m1.) 130 2, 200 2, 5002,500

25" l.- of' the broth was filteredby theaidof Hyde S'iqielj- Gel andt-hefiltrate was-passed-oven Am bei'lite (I-I'+ type) resin: The acidicfraction-Was then adsorbe don Dowex- K13177168- resin andeluted with 1-N ammonium hydroxide.- The: eluate was concentrated in vacuoanddecolorizedwith activated carbon: The pH was adjustedto. about 1.5 byfurther treatmentwith Amberlite IR-g200 1 resin" and then calciumhydroxide was added to .pH-of 6.0-"'=6.5. Aften filtering; pH of the;filtrate was again. adjusted to v 1.5 with- Amberlite IR-200 resin. Then2- ketoeL-gulonie acid was adsorbed on Dowex' X-E=l 68 resin and elutedwith"0.1 N4 ammonium hydroxide. The: eluatew-asaconoentratedin-vacuo andthen g-eof CI'ySjtallineZ-ketmL-gtilonicacidwas obtaincd. The physicabcharacteristics of this crystallineproduct were identical with those ofauthentic Z-keto-L-gulonic acid.

Example" 6 The following: organisms were incubated as descrihedinExample4; and the productionfof- Z-RetO-L-gulo'nie acid was:ascertained by paperchxomatog'raphy with-the solvent system phenolsaturated with-Water:

Acztb'liacter suboxydans, NRRL B-72 A'cetobacter glucanicus; 9324* Acetobacter-xylinum; ATCG 10245 At'etobacter albidus; IFO3250-Acetobacter a lbidus; IP9 325 1 A'cetObacteral-bidus; IFG- 325 3Acetobacterswboxydans, IFQ 325-5- Ace-tobact'er industrius 3260- Bacterium orleanense. 11306259 Acetobactercerinus, IFO 3 263- Acetobactencerinus,.-I1FO 32 64 Acetob actercerinus, .IFO 3265 I n Acetobacter.cerinus var. ammoniacus, IEO 3267 Acetob ac'ter cerinus var. an'moniacus, IFO 326.9- Acetobactefcerin'us, IFO' 3268 2-keto-L-gul0nicacid e/ml.) 500 1,600

8am Example. 8

A sele'cted strain of Pseudbmonas: striafaciens,IP0

3309; which wa's' used in' Example7 wasainenhatedfidescribed in? Examp1e5, substituting the; followingamedia for the media; used in:Example 5;

During cultivation: a sample'-0f-- the culture: broth was taken out fromthe fermentor atalternateday intervals and the amount ofi Z kCtOL-gUIODlG acid: was-estimated by themethod' described idExample 4.;

Result 1 obtainedas follows:

Days 3 5 5 I 7 2-keto-L-gulonic acid g/m1). 1, 500 f 3,600

' 53 g. of cryst'alline z -kew-L- mohic acid-was obtained in the sameWay asjEx'almple 5. The physical characteristicsof the crystallineprodn'ct were ideriticallwitli'ithose of authentic; 2-ketb-Lgulonicacid.

Example 9 One loopful of cells of Pseudomonas striafaciens, ,IiFO

3309; were inoculated with? a shake flaskicontain'ing theincu-batedon-arotary shaker at following medium and An'd the-prodilctionofZke'todl-igu-lbhic acid wae-asc'er.-

One loopful of cells of a selected strain of Acetobacter melqnogenz s,.ATCC 15163, was inoculated-intoa shake medium and incubated at} 30 C.with shaking for .18 hours:

Peic'en't Sorbitol 2.0 Glucose 0.5 Yeast-extract .0.5

A basal medium wasprepared for actual production of 2-keto-L gulonicacidi- 'Ih'ismedium 'h'ad the'followin-g co mpos'itionz .PIb6nilSor-bitol 5.0 Glucose V V 0.5 Ye'astextract 0.5 CaCO 2.0

500 ml, of the. incubated inoculum-wasadded? to-3'0' l; of the abovemediumvin astainless. fermentor-having a total volume of 50 l.Incubation was conducted at 28- 29 C. with stirring at the rate of 280r.p.m. with aeration atthe rate of 15-24 liters per minute, and withinner pressure at 15-20 lbs. per square inch. After 150 hoursincubation, the product, Z-keto-L-gulonic acid, was attained to about650 mg. per 100 ml. in the broth. Cells and solid fraction of the brothwere discarded by a Sharples type centrifuge and about 27 liters of theclear filtrate were obtained. 1 liter of the filtrate was decolorizedwith 50 g. of activated carbon and passed over Amberlite IR-120 resinand then concentrated in vacuo. The yield of crystalline2-keto-L-gulonic acid was about 5.3 g. per liter of the filtrate.

Example 11 One loopful of cells of a strain of the Pseudomonas sp., ATCC15165, was inoculated into a shake flask containing 500 mi. of thefollowing aqueous inoculum medium and incubated at 30 C. with shakingfor 18 hours:

Percent Sorbitol 2.0 Glucose 0.5 Yeast extract 0.5

A basal medium was prepared for production of 2-keto- L-gulonic acid.The medium had the following composition:

Percent Sorbitol 5.0 Glucose 0.5 Yeast extract 0.5 CaCO 2.0

500 ml. of the incubated inoculum was added to 30 l. of the above mediumin a stainless fermentor having a total volume of 50 l. Incubation wasconducted at 28-29 C. with stirring at the rate of 280 r.p.m., withaeration atv the rate of l5-24 liters per minute, and with innerpressure of -20 lbs. per square inch. After 150 hours incubation, theproduct, 2-keto-L-gulonic acid Was attained in an amount of about 120mg. per 100 ml. in the broth. Cells and solid fraction of the broth werediscarded by a- Sharples type centrifuge and about 27 liters of theclear filtrate were obtained. 1 liter of the filtrate was decolorizedwith 50 g. of activated carbon and passed over Amberlite IR-l20 resinand then concentrated in vacuo. The yield of crystals of2-keto-L-gulonic acid was about 0.97 g. per liter of the filtrate.

Example 12 One loopful of cells of a selected strain of Acetobacter sp.,ATCC 15164, were inoculated into a shake flask containing 500 ml. of thefollowing inoculum medium and incubated at 30 C. with shaking for 18hours:

Percent Sorbitol 2.0 Glucose 0.5 Yeast extract a 0.5

A basal medium was prepared for production of 2-keto- L-gluconic acid.The medium had the following composition:

Percent Sorbitol 5.0 Glucose 0.5 Yeast extract 0.5 CaCO 2.0

500 ml. of the incubated inoculum was added to 30 1. of the above mediumin a fermentor having a total volume of 50 l. Incubation was conductedat 2829 C. with stirring at the rate of 2:80 r.p.m., with aeration atthe rate of 15-24 liters per minute, and with inner pressure at l5-20lbs. per square inch. After 150 hours incubation, a half amount of theculture broth was passed over Amberlite IR-120 (H-type) resin,decolorized with activated carbon and then concentrated. The resulting10 product was filtered out, and after washing with acetone, 60 .g. ofcrystalline product were obtained.

(1) To 30 g. of the crystals thus obtained, 240 ml. of methanol and 0.3ml. of 98% sulfuric acid were added. After the mixture was heated underagitation for three hours, methanol was removed. The residue was washedwith a small amount of methanol and dried. The obtained product in 108ml. of methanol was refluxed with about 8 g. of sodium methylate underheating. After cooling, the resulting crystals were filtered out andwashed with a small amount of methanol to give 26.3 g. of sodium salt ofL-ascorbic acid. 2 2.5 g. of L-ascorbic acid were obtained from thesodium salt of L-ascorbic acid.

(2) A mixture of 30 g. of the crystalline product obtained from theculture broth, 240 ml. of methanol and 6 g. of Amberlite IR-2-O0 resinwere heated under agitation for three hours. After the reaction, theresin was filtered off. Methanol was distilled off from the filtrate andthe residue was Washed with a small amount of methanol. Then, 22.5 g. ofL-ascorbic acid were obtained in the same way as in the procedure (1)mentioned above.

Example 13 The residual half amount of the culture broth obtained inExample 12 was decolorized with activated carbon and the activatedcarbon was filtered off. The filtrate was passed over Amberlite IR(II-type) and was dried under reduced pressure. The dried product wasdissolved in 700 ml. of methanol. The solution was filtered after thetreatment with 5.2 g. of activated carbon. To the filtrate, 0.9 ml. of98% sulfuric acid was added. The mixture was heated under agitation forthree hours. Then methanol was distilled off from the reaction mixture.The residue was washed with a small amount of methanol and then dried.Then in the same way as in the procedure (2) in Example 12, 22.5 .g. ofL-ascorbic acid was obtained. The same result was also obtained in thesame Way as in the procedure (2) in Example 12 except using 21 g. ofAmberlite-200 in place of 0.9 ml. of 98% sulfuric acid.

Example 14 Cells of a selected strain of Acetobacter cerinus, grown onsorbitol-yeast extract agar slant, for 2 days at 28 C. were suspended in10 ml. of sterile water. 5 ml. of this suspension was inoculated into 1I. shake flask containing ml. of the following aqueous medium,previously sterilized in an autoclave at 15 lbs. per square inch steampressure:

Percent Sorbitol 5.0 Glucose 0.5 Yeast extract 0.5 CaCO 2.0

Incubation was maintained at 28 C. with shaking at the rate of 22-5r.p.-m. for 10 days. After the culture broth was collected and treatedwith Amberlite IRA-400, it was concentrated and cooled. The resultingcrystals were washed with acetone and then dried. 0.1 ml. of n butanol,0.45 ml. of water, 0.5 ml. of 35% hydrochloric acid and 18 ml. ofbenzene were added to the obtained crystals and the mixture was stirredat 64 C. for 16 hours. After the reaction, the resulting L-ascorbic acidWas filtered out. The crude L-ascorbic acid was washed with a mixture ofethanol and benzene, and then recrystallized to give 5.2 g. of pureL-ascorbic acid.

Example 15 Cells of a mutant strain of Pseudomonas striafaciens wereinoculated into a 2 1. shake flask containing 500 ml. of the followingaqueous inoculum medium and incubated on a rotary shaker at 28 C.:

Percent Sorbitol 2.0

Yeast extract 0.5

about '15 g. of sodium ethylate for 100 minutes. cooling, theresultingcrystals were filtered out to give 37.2

Incubation was maintained at 28. C. with shaking .atthe.

rate of 200 r.p.m. for 24 hours. A basal medium was The medium had thefoliowing-composition:

500 ml. of the incubated inoculum Was added to 30.1 of the-above mediumin a fei lnentor having a total volume 50 l. at the rate of 260 r.p.m.and with aeration at the rate of 30 l. per minutefor 7 days to obtain 27l. of culture broth. The culture broth was filtered and the filtrate wasto give 425 g. of the concentrated culture broth.

(1). A mixture of 250 g. of the concentrated culture ml. of 98% sulfuricacid was heated under agitation.

During the reaction, an azeotropic mixture consisting of prepared foractual production of .2-keto-L-gulonic acid.

Incubation Was conducted at 28 C. with stirring treated with AmberilteIR-l under reduced pressure water, ethanol and benzene was distilledoff. The-distillate. separated. into two layers and Water of the lower.layer was taken oif from reaction system and the upper; After the.reaction, solvents Weredistilled off and the residue was layer Wasbrought into the reaction system.

washed'with a small amountof ethanol and dried- The obtained product in160 ml. of ethanol .was refluxed with g. of sodium salt of L-ascorbic.acid. 32 g. of L-ascorbic acid was obtained fromthe sodium salt ofL-ascorbicacid. (2) A mixture of 175 g. of the concentrated culturebroth mentioned above and 5.3 ml. of 35% hydrochloric acidwasheated at75-80" C. for 5 hours. After the reac After tion mixture wasdecolorized, it was concentrated and dried at low temperature,preferably, to give crude L- ascorbicacid. The crude L-ascorbic acid wasrecrystallizedwith a small amount of Waterto give 7 g. of pureL-ascorbic acid.

Example 16 Cells of a mutant strain of Pseudomonas coronafaciens wereinoculated in a similar way as in Example 15 to prepare 26 1. of culturebroth- Thus prepared culture broth was treated ina similar way as theprocedure .(l) in Example '12 to obtain 58.5 g. of L-ascorbic acid.

Example 17 Cells of the following strains were incubated in a similarway as in Example-14." The-obtained-culture broth was treated in asimilar 'way as in Example 12 to give L-ascorbic acid.

Although the present invention has been described in conjunction withpreferred embodiments, it is to be understood that modifications andvariations may be resorted to without departing from the spirit andscope of the invention, as those skilled in the art will readilyunderstand.

broth, 600 ml. of ethanol, 300ml. of benzene and 0.6

Such modifications andvariations areconsidered to be within the purviewand scope of the invention and --ap-;

pended "claims. Having thus disclosed .the invention, :What is claimedis: 1. A .method for producing 2-keto-L-gulonic acid, which comprisesenzymatically transforming sorbitol di-I. rectly to Z-keto-L-gulonicacid 'by the action of the enzymesystemofza microorganism .capable ofconverting sorbitol to .Z-keto-L-gulonic acid andnselected from thefamily Pseudomonadaceae, and, recovering the 'so1produced2-keto-L-gulonic acid.

2. A method for producing .2-keto-L-gulo't1ic. acid,-.

which comprises enzymatically transforming sorbitoldi-I rectly to2-keto-L-gulonic acid 'by the action of the enzyme system of amicroorganism capable of converting v sorbitol to -2-keto-L-gulonic.acid andaselected from the group including the: genus .Pseudomonas-andthe genus Acetobacter, and n'ecovering "the so-produce;d:2-.keto-L-gulonic acid.

3. A .method for producing :2-keto-L-gulonic acid, which comprisesenzymatically transforming sorbitol di-' rectly to 2-keto-L+gulonic acidby the action of the enzyme system of a microorganism capabletofconverting sorbitol to 2-keto-L-gulonic acid and selected from thegenus Pseudomonas, and recovering the :so-produced 2-I 'keto-L-gulonicacid.

4. A .method for. producing Z-keto-L-gulonic acid, which comprisesenzymatically transforming sorbitol di rectlyto "2-keto-L+gulonic:acidby the action of the enzyme system of a microorganism capable'ofconverting sorbitol to 2-keto-L-gulonic acid and selected from the genusAcetobactcr, .and recovering-the so-produced 2- keto-L-gulonicacid.

5. The method of claim 3,-wherein the. microorganism.

is Pseudomonas sp., ATCC 15165.

6.= The method ofclaim 4, =wherein' the microorganism is a strain .ofAcetobacter melanogenum.

7. The method o'f claim 4, wherein theqmicroorganism is Acetobactersp,.ACTT 15164.

8; The method of claim 4, wherein the. microorganism is a strain ofAcetobacter cerinus.

9. The method of claim 4, :wherein the! microorganism is a strainofAcetobacter.suboxydans.

10. Themethocl of claim 4, wherein :the microorganism is a strain ofAcetobactengluconicus.

11. The method of claim 4, wherein the microorganism is a strain ofAcetobacter xylinum.

12.' The method of claim4, wherein the microorganism is a strain ofAcezobacter albidus.

13. The method of claim 4, wherein the microorganism is a strain ofAcetobaczer industriumt.

14. A method: for producirig Lrascorbic acid, which comprisestransforming sorbitol to 2-.keto-L-gulonic acid by the action of theenzyme system of'a microorganism selected from the group includingthegenus 'Pseudomonas and the genus Acetobacter, followed bysubjecting-there. suiting 2-keto-L-gulonic acid to esterification',enolization and .lactonization.

15. A method for the enzymatic production of 2-keto- L-gulonic ,aciddirectly from sorbitol which comprises subjecting the sorbitol to theaction of the enzyme system produced by culturing a microorganismselected from the group consisting of Acetobacler melanogenus, ATCC15163 (I FO 3292) Acetobactersp, ATCC 15164: Acetobacter rubiginosus,IFO 3243 Ace'tobacter suboxydans, NRRL B,72 Acetobacter gluconicus, ATCC'93 24.

Acetobac'ter xylinum, ATCC 10245 Acetobacter albidus, IEO 3250Acetobacteralbidus, IFO 3251 Acetobacter albidusylFO .3253

Acetobacter suboxydans, IFO.3255 4 Acetobacter industrz'us, IFO 3260Bacterium orleanense, IFO 3259 Acetobacter cerinus, IFO 3263 Acetobactercerinus, IFO 3264 Acetobacter cerinus, IFO 3265 Acetobacter cerinus, [F3266 Acetobacter cerinus var. ammoniacus, IFO 3267 Acetobacter cerinusvar. ammoniacus, IFO 3269 and Acetobacter cerinus, IFO 3268 in a culturemedium therefor containing the sorbitol, whereby the latter is directlyconverted into 2- keto-L- gulonic acid which accumulates in the saidmedium, and recovering thus-accumulated Z-keto-L-gulonic acid from thelatter.

16. A method for the enzymatic production of 2-keto- L-gulonic aciddirectly from sorbitol which comprises subjecting the sorbitol to theaction of the enzyme systern produced by culturing a microorganismselected from the group consisting of Pseudomonas striafaciens, IFO 3309Pseudomonas coronafaciens, IFO 3310 and Pseudomonas sp., ATCC 15165 in aculture medium therefor containing the sorbitol, whereby the latter isdirectly converted into 2-keto-L- gulonic acid which accumulates in thesaid medium, and recovering thus-accumulated 2-keto-L-gulonic acid fromthe latter.

17. A method for the enzymatic production of 2-keto- L-gulonic aciddirectly from sorbitol which comprises subjecting the sorbitol to theaction of the enzyme system produced by culturing Pseudomonas sp., ATCC151.65, in a culture medium therefor containing the sorbitol, wherebythe latter is directly converted into Z-keto-L-gulonic acid whichaccumulates in the said medium, and recov- 'ering thus-accumulatedZ-keto-L-gulonic acid from the latter.

18. A method for the enzymatic production of 2- keto-L-gulonic aciddirectly from sorbitol which comprises subjecting the sorbitol to theaction of the enzyme system produced by culturing Acetobacter sp., ATCC15164, in a culture medium therefor containing the sorbitol, whereby thelatter is directly converted into 2- keto-L-gulonic acid whichaccumulates in the said medium, and recovering thus-accumulated2-keto-L- gulonic acid from the latter.

19. A method for the enzymatic production of Z-keto- L-gulonic aciddirectly from sorbitol which comprises subjecting the sorbitol to theaction of the enzyme system produced by culturing Acetobactermelanogenus, ATCC 15163, in a culture medium therefor containing thesorbitol, whereby the latter is directly converted into 2-keto-L-gallonic acid which accumulates in the said medium, and recoveringthus-accumulated 2-keto-L-gulonic acid from the latter.

20. A method for the preparation of 2-keto-L-gulonic acid whichcomprises pre-culturing a 2-keto-L-gulonicproducing microorganismselected from the group consisting of the genus Pseudomonas and thegenus Acetobacter, in an aqueous inoculum medium containing sorbitol,inoculating the thus-produced inoculum into a sorbitolcontaining medium,culturing the microorganism in the latter, whereby an enzyme system isproduced which directly converts the sorbitol into 2-keto-L-gulonic acidwhich accumulates in the last-named medium, and recoveringthus-accumulated 2-keto-L-gulonic acid from the latter.

Prescott et al.: Industrial Microbiology, 3rd ed., 1959, pp. 456-458.

A. LOUIS MONACELL, Primary Examiner.

L. M. SHAPIRO, Assistant Examiner.

1. A METHOD FOR PRODUCING 2-KETO-L-GULONIC ACID, WHICH COMPRISESENZYMATICLLY TRANSFORMING SORBITOL DIRECTLY TO 2-KETO-L-GULONIC CID BYTHE ACTION OF THE ENZYME SYSTEM OF A MICROORGANISM CAPABLE OF CONVERTINGSORBITOL TO 2-KETO-L-GULONIC ACID AND SELECTED FROM THE FAMILYPSEUDOMONADACEAE, AND RECOVERING. THE SO-PRODUCED 2-KETO-L-GULONIC ACID.